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WO1999012935A1 - Derives de pyrrolopyrrolidine et leur utilisation comme inhibiteurs de serine protease - Google Patents

Derives de pyrrolopyrrolidine et leur utilisation comme inhibiteurs de serine protease Download PDF

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Publication number
WO1999012935A1
WO1999012935A1 PCT/EP1998/005744 EP9805744W WO9912935A1 WO 1999012935 A1 WO1999012935 A1 WO 1999012935A1 EP 9805744 W EP9805744 W EP 9805744W WO 9912935 A1 WO9912935 A1 WO 9912935A1
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Prior art keywords
inhibitor
enzyme
library
serine protease
compound
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PCT/EP1998/005744
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English (en)
Inventor
Steven John Coote
Michael Dennis Dowle
Harry Finch
Michael Menteith Hann
Henry Anderson Kelly
Simon John Fawcett Macdonald
Neil Anthony Pegg
Nigel Grahame Ramsden
Nigel Stephen Watson
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Glaxo Group Limited
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Priority to JP2000510742A priority Critical patent/JP2001515906A/ja
Priority to EP98950011A priority patent/EP1015456A1/fr
Priority to AU96243/98A priority patent/AU9624398A/en
Publication of WO1999012935A1 publication Critical patent/WO1999012935A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D487/00—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00
    • C07D487/02—Heterocyclic compounds containing nitrogen atoms as the only ring hetero atoms in the condensed system, not provided for by groups C07D451/00 - C07D477/00 in which the condensed system contains two hetero rings
    • C07D487/04—Ortho-condensed systems
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system
    • A61P11/06—Antiasthmatics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
    • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
    • C—CHEMISTRY; METALLURGY
    • C40—COMBINATORIAL TECHNOLOGY
    • C40B—COMBINATORIAL CHEMISTRY; LIBRARIES, e.g. CHEMICAL LIBRARIES
    • C40B40/00—Libraries per se, e.g. arrays, mixtures

Definitions

  • This invention relates to a new class of chemical compounds and their use as inhibitors of serine protease enzymes. These compounds are useful as pharmaceuticals and we provide processes for preparing them and formulations containing them.
  • Serine proteases are a class of proteolytic enzymes characterised by having at the active site a serine residue which interacts with the carbonyl carbon of a peptide bond to cleave the peptide bond via an acyl enzyme intermediate.
  • the active site serine is generally numbered Ser-195.
  • Most members of the family of serine proteases have a histidine and an aspartic acid residue in the active site (numbered His-57 and Asp-102 based on chymotrypsin) which activate the serine hydroxyl group to attack the scissile peptide carbonyl.
  • the mechanism of hydrolysis of peptide bonds by serine proteases is believed to be similar for all enzymes in the family, it is well known that their substrate specificities differ dramatically.
  • specificity is shown for peptide bonds which have a particular moiety ⁇ to the scissile peptide carbonyl which in conventional nomenclature is said to be in the P 1 position and to occupy the S 1 specificity subsite (see Schlecter and Berger (1967) Biochem Biophys Res Common 27 157).
  • the preferred substrate for thrombin is a peptide containing a basic residue (e.g. arginine i.e.
  • Serine proteases are widespread in the human body and abnormal or excessive activity of serine proteases is implicated in a diverse range of diseases and conditions (see “Proteinase Inhibitors", Barrett and Salveson (1986), Elsevier, p56; Drugs Future (1996), 21(8), 811-816; Exp. Opin. Ther. Patents (1997) 7(1) 17-28).
  • Neutrophil elastase is found in neutrophil azurophilic granules associated with tissue inflammation and is associated with a number of inflammatory diseases including emphysema, chronic bronchitis and adult respiratory distress syndrome (ARDS).
  • ARDS adult respiratory distress syndrome
  • thrombin Members of the blood coagulation cascade (e.g. thrombin, Factor Vila, Factor Xa, Factor Xla, Factor Xlla) and members of the fibrinolytic cascade (e.g. tissue plasminogen activator and plasmin) are potential targets for treatment of diseases of the vascular system.
  • thrombin is a potential target for the treatment of thrombosis.
  • Tissue plasminogen activator and plasmin may also be implicated in tumour metastasis.
  • Tryptase is present in mast cells and inhibitors of tryptase have shown efficacy in models of asthma.
  • Pancreatic elastase, trypsin and chymotrypsin are associated with digestive disorders such as pancreatitis.
  • Cathepsin G is associated with emphysema.
  • Serine proteases are also widespread in human pathogens especially viruses and these provide an attractive target for the treatment of pathogenic diseases and conditions.
  • Herpes viruses encode a serine protease which is crucial for viral replication and is therefore a target for the treatment of conditions caused by these viruses.
  • Herpes family of viruses is responsible for a wide range of human infectious diseases including chicken pox and shingles (varicella and Herpes zoster viruses, respectively), cold sores and genital herpes (herpes simplex virus), retinitis, pneumonitis and keratitis (human cytomegalovirus, hCMV), as well as diseases caused by Epstein Barr Virus (EBV), human herpes virus 6 (HHV 6),
  • EBV Epstein Barr Virus
  • HHV 6 human herpes virus 6
  • inhibitors of serine protease enzymes which are substituted derivatives of trans- hexahydropyrrolo[3,4-b]pyrrol-2-one.
  • this invention relates to inhibitors of serine protease enzymes which are compounds of formula I:
  • R 1 is a moiety adapted to fit in the Si specificity subsite of the enzyme
  • R 2 is a moiety adapted to optimise the potency, pharmacokinetics, pharmacodynamics, selectivity and enzyme kinetic properties of the inhibitor
  • R 3 is a moiety adapted to optimise the potency, pharmacokinetics, pharmacodynamics, selectivity and physicochemical properties of the inhibitor; and physiologically acceptable salts and solvates thereof.
  • translactam template of formula I is highly complementary to the active site of serine proteases and the lactam carbonyl mimics the peptide carbonyl of the enzyme's natural substrate.
  • Inhibition of serine proteases by compounds of the invention has been found to be either competitive (reversible) or time-dependent (acylating) depending on the precise enzyme and particular substitution pattern on the translactam template.
  • Time-dependent (acylating) inhibition is believed to occur when attack of the enzyme active site serine on the translactam carbonyl causes opening of the strained lactam ring generating an enzyme acylated at the serine sidechain.
  • the advantages of our invention reside inter alia in that (a) the trans- hexahydropyrrolo[3,4-b]pyrrol-2-one template is completely new and therefore highly desirable in a medicament especially for the treatment of pathogenic conditions which are prone to drug resistance, (b) the trans- hexahydropyrrolo[3,4-b]pyrrol-2-one template may be highly functionalised and is therefore ideal for the specific and selective inhibition of a wide range of different enzymes, (c) the trans-hexahydropyrrolo[3,4-b]pyrrol-2-one template may potentially be functionalised to give (i) high or low metabolic stability and (ii) competitive or time-dependent inhibition as desired.
  • the determination of the optimum substitution of the derivatives of trans- hexahydropyrrolo[3,4-b]pyrrol-2-one, especially regarding selection of groups R 1 , R 2 and R 3 for a particular serine protease enzyme can be made in a conventional manner, namely: (a) by preparation of a number of compounds having sufficient diversity especially in groups R 1 , R 2 and R 3 , (b) treatment of a sample of the enzyme in question with a sample of each of the compounds so prepared and (c) determining the extent to which inhibition of the enzyme has occurred.
  • Suitable R 1 groups will fit appropriately in the Si specificity subsite of the target enzyme.
  • Choice of group R 1 may be made having regard to the known substrate specificity preferences of the target enzyme, crystallographic information concerning the geometry of the Si specificity subsite of the target enzyme and/or empirical determination based on screening data (see for example "Proteinase Inhibitors” Barrett and Salveson (1986), Elsevier, p9 and p59).
  • the group R 1 is preferably small and hydrophobic, e.g. C 2-4 alkyl or C 2-4 alkenyl, especially propyl or isopropyl, particularly isopropyl.
  • the group R 1 is preferably large and hydrophobic, e.g. (CH 2 ) 1-2 Ph, (CH 2 ) 0 - 2 cyclohexyl, t-butyl.
  • Ph represents phenyl or substituted phenyl (e.g. phenyl substituted by C 1 __ 6 alkyl, halogen).
  • Planar aromatic sidechains e.g. benzyl are especially preferred.
  • R 2 will be a moiety adapted to optimise the potency, pharmacokinetics, pharmacodynamics, selectivity and enzyme kinetic properties of the serine protease inhibitor.
  • R 2 will be lactam activating moiety.
  • Suitable activating groups include electron withdrawing groups which may typically (but not exclusively) comprise a SO 2 or CO moiety attached to the lactam nitrogen.
  • R 2 may represent CHO or SO ⁇ alkyl and is preferably a group -SO 2 Me.
  • R 2 is preferably a group CONH(CH 2 ) 1-4 Ph, SO 2 (CH 2 ) 0 ..,Ph.
  • -COOC ⁇ alkyl e.g. -COOMe
  • -CONHC 1-4 alkyl e.g. -CONHMe
  • R 2 When R 2 is highly activating we find that the inhibitors act through a time- dependent (acylating) mechanism whereas when R 2 is less activating, the inhibitors may act through a reversible (competitive) mechanism.
  • R 2 comprises an SO 2 moiety attached to the lactam nitrogen
  • the inhibitor is generally time-dependent (acylating).
  • R 2 comprises a CO moiety attached to the lactam nitrogen
  • the inhibitor may be time-dependent (acylating) or not depending on the exact nature of R 2 .
  • R 2 represents it is more likely to be time-dependent (acylating) than when R 2 represents CONHC ⁇ alkyl.
  • R 3 will be a moiety adapted to optimise the potency, pharmacokinetics, pharmacodynamics, selectivity and physicochemical properties of the serine protease inhibitor. It may also be adapted to optimise other pharmacological properties such as water solubility and oral activity (if desired).
  • R 3 can vary quite widely and a person skilled in the art would be able to determine from suitable testing if a given R 3 is suitable for the aforementioned purposes or not.
  • R 3 comprises a CO, SO 2 or CO.O (especially a CO or SO 2 ) moiety attached directly to the pyrrolidine nitrogen and is, for example, a group of formula R 30 CO, R 30 SO 2 or R 30 OCO (especially R 30 CO or R 30 SO 2 ).
  • R 30 will also be a moiety adapted to optimise the potency, pharmacokinetics, pharmacodynamics, selectivity and physicochemical properties of the serine protease inhibitor and may represent, for example, alkyl (e.g. C 1-8 alkyl), alkenyl (e.g. C 1-8 alkenyl), aryl, alkylaryl (e.g. C 1-8 alkylaryl), or alkenylaryl (e.g. C ⁇ 8 alkenylaryl).
  • alkyl e.g. C 1-8 alkyl
  • alkenyl e.g. C 1-8 alkenyl
  • aryl e.g. C 1-8 alkylaryl
  • alkenylaryl e.g. C ⁇ 8 alkenylaryl
  • alkyl includes branched and cyclic alkyl.
  • Alkenyl includes branched and cyclic alkenyl.
  • Aryl includes mono and bicyclic aromatic rings optionally containing heteroatoms, e.g. O, N and S atoms (for example 1 to 4 heteroatoms).
  • Amine groups include primary, secondary and tertiary amine groups including cyclic amine.
  • the extent to which inhibition has occurred may be determined by conventional assay techniques including (but not limited to) chromogenic assays, fluorogenic assays, HPLC and scintillation proximity assays.
  • a library comprising a plurality of substituted derivatives of trans-hexahydropyrrolo[3,4-b]pyrrolo-2- one will be prepared.
  • the library will comprise a plurality of compounds of formula I
  • R 1 is a moiety adapted to fit in the Si specificity subsite of the enzyme
  • R 2 is a moiety adapted to optimise the potency, pharmacokinetics, pharmacodynamics, selectivity and enzyme kinetic properties of the inhibitor
  • R 3 is a moiety adapted to optimise the potency, pharmacokinetics, pharmacodynamics, selectivity and physicochemical properties of the inhibitor; and physiologically acceptable salts and solvates thereof.
  • the library will, ideally comprise at least 10 (e.g. 10, 100, 1000 or more) different compounds.
  • a library of compounds of formula I wherein R 1 represents a small and hydrophobic group e.g. C 2-4 alkyl or C 2-4 alkenyl, especially propyl or isopropyl, particularly isopropyl may be particularly useful for screening for an inhibitor of elastase-like enzymes e.g. neutrophil elastase.
  • a library of compounds of formula I wherein R 1 represents a large and hydrophobic group e.g. (CH 2 ) ⁇ -2 Ph, (CH 2 ) 0-2 cyclohexyl or t-butyl may be useful for screening for an inhibitor of a chymotrypsin-like enzyme e.g. chymotrypsin or cathepsin G.
  • the library may be a solid phase or a solution phase library. It may be a discrete library or a pooled library.
  • R 1 is a moiety adapted to fit in the Si specificity subsite of the enzyme; or a protected derivative thereof, by sequential reaction to introduce the desired
  • a metal catalyst e.g. palladium supported on carbon
  • the compound of formula III may be prepared by the method of Archille Barco et a/ (1992), J. Org. Chem., 57, 6279.
  • a base e.g. triethylamine
  • an inert solvent such as acetonitrile or dichloromethane.
  • a metal catalyst e.g. palladium supported on carbon
  • a base e.g. triethylamine
  • an inert solvent such as acetonitrile or dichloromethane.
  • Step v This is generally an alkylation reaction which may be achieved by the sequential reactions with a strong base, e.g. LHMDS, in the presence of DMPU in an inert solvent such as THF or diethyl ether at -70°C, followed by alkyl halide, e.g. bromide or iodide, at -70°C -room temperature.
  • a strong base e.g. LHMDS
  • alkyl halide e.g. bromide or iodide
  • an acid e.g. trifluoroacetic acid or hydrogen chloride
  • a suitable solvent such as dichloromethane or dioxane.
  • This cyclisation may be achieved using t-butyl magnesium chloride in the presence of TMEDA in an inert solvent such as THF or diethyl ether.
  • TMEDA inert solvent
  • the use of TMEDA is optional.
  • this cyclisation may be performed by first hydrolysing the carboxylic acid ester (e.g. with sodium hydroxide) and then cyclising the resultant acid using diphenylphosphoryl azide in the presence of triethylamine in an inert solvent such as DMF or THF.
  • This conventional deprotection reaction can be achieved by reacting with hydrogen in the presence of a metal catalyst, e.g. palladium supported on carbon, and a proton source, e.g. ethereal hydrogen chloride, in a suitable solvent such as ethyl acetate or ethanol.
  • a metal catalyst e.g. palladium supported on carbon
  • a proton source e.g. ethereal hydrogen chloride
  • a suitable solvent such as ethyl acetate or ethanol.
  • the use of a proton source is optional.
  • the anion of the compound of formula (VII) is first prepared by treatment with a strong base, e.g. LHMDS, followed by treatment with acetone.
  • a strong base e.g. LHMDS
  • Standard dehydration conditions may be used for this reaction, e.g. treatment with concentrated sulphuric acid. These conditions will remove the BOC deprotecting group, otherwise the deprotection can be performed as an additional step.
  • This oxidation may be suitably carried out under nitrogen using Swern oxidation conditions, e.g. oxalyl chloride and DMSO at -80°-0° in dichloromethane, followed by triethylamine.
  • Swern oxidation conditions e.g. oxalyl chloride and DMSO at -80°-0° in dichloromethane, followed by triethylamine.
  • Tebbe reaction conditions e.g. ⁇ - cchhlloorroobbiiss(( ⁇ 55 --22,,44--ccyyccllooppeennttaaddiieenn--11--yyll))(dimethylaluminium)- ⁇ -methylenetitanium in THF at around 0°- room temperature
  • This standard deprotection may be performed using an acid, e.g. trifluroacetic acid or hydorogen chloride , in a suitable solvent such as dichloromethane or dioxane.
  • an acid e.g. trifluroacetic acid or hydorogen chloride
  • a suitable solvent such as dichloromethane or dioxane.
  • Scheme 3 may also be adapted to produce compounds of formula (II) having other branched R 2 alkyl or alkenyl sidechains.
  • Enantiomers having the absolute stereochemistry shown in formula (la) are especially preferred.
  • Enantiomerically pure compounds may be prepared by chiral separation or by synthesis based on chiral starting materials.
  • Suitable physiologically acceptable salts include inorganic base salts such as alkali metal salts (for example sodium and potassium salts) and ammonium salts and organic base salts.
  • Suitable organic base salts include amine salts such as trialkylamine (e.g. triethylamine), dialkylamine (e.g. dicyclohexylamine), optionally substituted benzylamine (e.g. phenylbenzylamine or p-bromobenzylamine), procaine, ethanolamine, diethanolamine, N-methylglucosamine and tri(hydroxymethyl)methylamine salts and amino acid salts (e.g. lysine and arginine salts).
  • Suitable inorganic and organic acid salts include the hydrochloride, trifluoroacetate and tartrate.
  • the compounds of the invention may be formulated for administration in any convenient way, and the invention therefore also includes within its scope pharmaceutical compositions for use in therapy, comprising a compound of the invention or a physiologically acceptable salt or solvate thereof in admixture with one or more physiologically acceptable diluents or carriers.
  • the compounds of the invention may, for example, be formulated for oral, buccal, parenteral, topical or rectal administration.
  • Tablets and capsules for oral administration may contain conventional excipients such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, mucilage of starch or polyvinyl pyrrolidone; fillers, for example, lactose, microcrystalline cellulose, sugar, maize- starch, calcium phosphate or sorbitol; lubricants, for example, magnesium stearate, stearic acid, talc, polyethylene glycol or silica; disintegrants, for example, potato starch, croscarmellose sodium or sodium starch glycollate; or wetting agents such as sodium lauryl sulphate.
  • the tablets may be coated according to methods well known in the art.
  • Oral liquid preparations may be in the form of, for example, aqueous or oily suspensions, solutions, emulsions, syrups or elixirs, or may be presented as a dry product for constitution with water or other suitable vehicle before use.
  • Such liquid preparations may contain conventional additives such as suspending agents, for example, sorbitol syrup, methyl cellulose, glucose/sugar syrup, gelatin, hydroxymethyl cellulose, carboxymethyl cellulose, aluminium stearate gel or hydrogenated edible fats; emulsifying agents, for example, lecithin, sorbitan mono-oleate or acacia; non-aqueous vehicles (which may include edible oils), for example almond oil, fractionated coconut oil, oily esters, propylene glycol or ethyl alcohol; or preservatives, for example, methyl or propyl £- hydroxybenzoates or sorbic acid.
  • the preparations may also contain buffer salts, flavouring, colouring and/or sweetening agents
  • compositions may take the form of tablets or lozenges formulated in conventional manner.
  • the compounds may also be formulated as suppositories, e.g. containing conventional suppository bases such as cocoa butter or other glycerides.
  • the compounds of the invention may also be formulated for parenteral administration by bolus injection or continuous infusion and may be presented in unit dose form, for instance as ampoules, vials, small volume infusions or pre- filled syringes, or in multi-dose containers with an added preservative.
  • the compositions may take such forms as solutions, suspensions, or emulsions in aqueous or non-aqueous vehicles, and may contain formulatory agents such as anti-oxidants, buffers, antimicrobial agents and/or toxicity adjusting agents.
  • the active ingredient may be in powder form for constitution with a suitable vehicle, e.g. sterile, pyrogen-free water, before use.
  • the dry solid presentation may be prepared by filling a sterile powder aseptically into individual sterile containers or by filling a sterile solution aseptically into each container and freeze-drying.
  • topical administration as used herein, we include administration by insufflation and inhalation. Examples of various types of preparation for topical administration include ointments, creams, lotions, powders, pessaries, sprays, aerosols, capsules or cartridges for use in an inhaler or insufflator or drops (e.g. eye or nose drops).
  • Ointments and creams may, for example, be formulated with an aqueous or oily base with the addition of suitable thickening and/or gelling agents and/or solvents.
  • bases may thus, for example, include water and/or an oil such as liquid paraffin or a vegetable oil such as arachis oil or castor oil or a solvent such as a polyethylene glycol.
  • Thickening agents which may be used include soft paraffin, aluminium stearate, cetostearyl alcohol, polyethylene glycols, microcrystalline wax and beeswax.
  • Lotions may be formulated with an aqueous or oily base and will in general also contain one or more emulsifying agents, stabilising agents, dispersing agents, suspending agents or thickening agents.
  • Powders for external application may be formed with the aid of any suitable powder base, for example, talc, lactose or starch. Drops may be formulated with an aqueous or non-aqueous base also comprising one or more dispersing agents, solubilising agents or suspending agents.
  • Spray compositions may be formulated, for example, as aqueous solutions or suspensions or as aerosols delivered from pressurised packs, with the use of a suitable propellant, e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, 1 ,1 ,1 ,2,3,3,3-heptafluoropropane, 1 ,1 ,1 ,2- tetrafluorethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g. dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, 1 ,1 ,1 ,2,3,3,3-heptafluoropropane, 1 ,1 ,1 ,2- tetrafluorethane, carbon dioxide or other suitable gas.
  • a suitable propellant e.g. dichlorodifluoromethane,
  • Compounds of the invention may also be used in purification and diagnostic applications involving serine protease enzymes.
  • an immobilised compound of the invention may allow a serine protease capable of binding that compound to be isolated.
  • a tagged compound of the invention may enable a serine protease capable of binding that compound to be identified.
  • enzyme activity is generally determined at a 15 minute timepoint. Enzyme kinetics may be investigated by determining enzyme activity at other timepoints (e.g. 0, 30 minutes).
  • the rate of increase in absorbance at 405nm is proportional to elastase activity.
  • Enzyme activity is plotted against concentration of inhibitor and an IC50 determined using curve fitting software.
  • Compounds of the invention may be tested for their thrombin inhibitory activity as determined in vitro by their ability to inhibit human ⁇ -thrombin in a chromogenic assay, using N-p-tosyl-Gly-Pro-Lys p-nitroaniiide as the chromogenic substrate. All dilutions were made in a buffer consisting of: 50mM HEPES, 150 mM NaCI, 5mM CaCI 2 , 0.1% PEG and at pH7.4. Briefly, the substrate (final cone, of 100 ⁇ M) was added to thrombin (final cone, of 1 nM) and the reaction monitored for 10mins at 405nm using a Biotek EL340 plate reader; the assay was performed at room temperature.
  • IC 50 values the data were analysed using Kineticalc ® with a 4-parameter curve fitting procedure to obtain the IC 50 value.
  • the compounds were preincubated with thrombin for these times prior to adding the chromogenic substrate.
  • hCMV serine protease used is a mutant of the 30K protease lacking the internal cleavage site (Ala142/Ala143) and which has been cloned in E.coli to produce active enzyme (hCMV ⁇ Ala protease).
  • IC 50 data for test compounds are determined after preincubation of the enzyme with test inhibitor compound for
  • Test compounds are dissolved in DMSO, serially diluted and added at a range of concentrations (from 100 ⁇ M - 0.195 ⁇ M) to a reaction containing 0.5 ⁇ M CMV ⁇ Ala protease, 100mM HEPES pH7.5, 0.2mM EDTA, 10mM NaCI, 1mM DTT, and 30% glycerol.
  • the reaction mixture is pre-incubated at 32°C for 15 minutes prior to addition of 4mM oligopeptide substrate (Arg-Glu-Ser-Tyr-Val-
  • Lys-Ala-pNA Lys-Ala-pNA
  • BIO-TEK Bio Kinetics Reader EL340i The plate reader monitors production of pNA and calculates the reaction rates over 30 minutes. The rates are plotted against inhibitor concentration and IC 50 values determined.
  • Compounds of the invention may be tested for their tryptase inhibitory activity as determined in vitro by their ability to inhibit human lung mast cell tryptase in a chromogenic assay, using N-p-Tosyl-Gly-Pro-Lys-p-nitroanilide as the chromogenic substrate.
  • Compounds were diluted from a 10mM stock solution in dimethylsulphoxide. All dilutions were made in a buffer consisting of: 10mM Tris- HCI, 120mM NaCI, pH 7.4. Briefly, the substrate (final cone, of 400 ⁇ M) was added to tryptase (final cone, of 0.11 ⁇ g.m.
  • Compounds of the invention may be tested for their trypsin inhibitory activity as determined in vitro by their ability to inhibit bovine trypsin in a chromogenic assay, using N-Benzoyl-lle-Glu-Gly-Arg-p-nitroanilide as the chromogenic substrate.
  • Compounds were diluted from a 10mM stock solution in dimethylsulphoxide. All dilutions were made in a buffer consisting of: 50mM Tris- HCI, 15mM CaCI 2 , pH 8.4.
  • the substrate final cone, of 160 ⁇ M
  • trypsin final cone, of 25ng.ml "1
  • compound at appropriate concentrations incubated for 15 minutes and the reaction monitored for 10 minutes at 405nm using a BioTek EL340 plate reader; the assay was performed at 37°C.
  • IC 50 values the data were analyzed using Kineticalc ® with a 4- parameter curve-fitting procedure.
  • Compounds of the invention may be tested for their Factor Xa inhibitory activity as determined in vitro by their ability to inhibit human Factor Xa in a chromogenic assay, using N- ⁇ -Benzyloxycarbonyl-D-Arg-Gly-Arg-p-nitroanilide as the chromogenic substrate.
  • Compounds were diluted from a 10mM stock solution in dimethylsulphoxide. All dilutions were made in a buffer consisting of: 50mM Tris-HCI, 150mM NaCI, 5mM CaCI 2 , pH 7.4.
  • the substrate final cone, of 200 ⁇ M
  • Factor Xa final cone, of 0.02 U.ml "1
  • compound at appropriate concentrations incubated for 15 minutes and the reaction monitored for 10 minutes at 405nm using a BioTek EL340 plate reader; the assay was performed at 37°C.
  • IC 50 values the data were analyzed using Kineticalc ® with a 4-parameter curve fitting procedure.
  • Compounds of the invention may be tested for their Factor XIa inhibitory activity as determined in vitro by their ability to inhibit human Factor XIa in a chromogenic assay, using L-Pyroglutamyl-Pro-Arg-p-nitroanilide as the chromogenic substrate.
  • Compounds were diluted from a 10mM stock solution in dimethylsulphoxide. All dilutions were made in a buffer consisting of: 8.1 mM NaH 2 PO 4 , 147mM KH 2 PO 4 , 2.7mM KCI, 137mM NaCI, pH 7.2.
  • the substrate final cone, of 400 ⁇ M
  • Factor XIa final cone, of 0.25 ⁇ g.ml "1
  • compound at appropriate concentrations incubated for 15 minutes and the reaction monitored for 10 minutes at 405nm using a BioTek EL340 plate reader; the assay was performed at 25°C.
  • IC 50 values the data were analyzed using Kineticalc ® with a 4-parameter curve fitting procedure.
  • Compounds of the invention may be tested for their Factor Xlla inhibitory activity as determined in vitro by their ability to inhibit human Factor Xlla in a chromogenic assay, using H-D-Pro-Phe-Arg-p-nitroanilide as the chromogenic substrate.
  • Compounds were diluted from a 10mM stock solution in dimethylsulphoxide. All dilutions were made in a buffer consisting of: 28mM NaBarbitone, 125mM NaCI, 1mM EDTA, pH 7.35. Briefly, the substrate (final cone, of 200 ⁇ M) was added to Factor Xlla (final cone, of 1.25 ⁇ g.m. "1 ) and compound at appropriate concentrations incubated for 15 minutes and the reaction monitored for 10 minutes at 405nm using a BioTek EL340 plate reader; the assay was performed at 25°C.
  • Compounds of the invention may be tested for their tissue plasminogen activator inhibitory activity as determined in vitro by their ability to inhibit human tissue plasminogen activator in a chromogenic assay, using MeSO 2 -D-CHT-Gly-Arg-p- nitroanilide as the chromogenic substrate.
  • Compounds were diluted from a 10mM stock solution in dimethylsulphoxide. All dilutions were made in a buffer consisting of: 50mM Tris-HCI, 150mM NaCI, pH 8.4.
  • the substrate final cone, of 750 ⁇ M
  • tissue plasminogen activator final cone, of 1.O ⁇ g.ml "1
  • compound at appropriate concentrations incubated for 15 minutes and the reaction monitored for 10 minutes at 405nm using a BioTek EL340 plate reader; the assay was performed at 30°C.
  • IC 50 values the data were analyzed using Kineticalc ® with a 4-parameter curve-fitting procedure.
  • Compounds of the invention may be tested for their plasmin inhibitory activity as determined in vitro by their ability to inhibit human plasmin in a chromogenic assay, using H-D-Val-Leu-Lys-p-nitroanilide as the chromogenic substrate.
  • Compounds were diluted from a 10mM stock solution in dimethylsulphoxide. All dilutions were made in a buffer consisting of: 50mM Tris-HCI, 150mM NaCI, 5mM CaCI 2 , pH 7.4.
  • the substrate final cone, of 363 ⁇ M
  • plasmin final cone, of 0.02 U.ml "1
  • compound at appropriate concentrations incubated for 15 minutes and the reaction monitored for 10 minutes at 405nm using a BioTek EL340 plate reader; the assay was performed at 37°C.
  • IC 50 values the data were analyzed using Kineticalc ® with a 4-parameter curve fitting procedure.
  • Compounds of the invention may be tested for their Factor Vila inhibitory activity as determined in vitro by their ability to inhibit human Factor Vila in a chromogenic assay, using H-D-lle-Pro-Arg-p-nitroanilide as the chromogenic substrate.
  • Compounds were diluted from a 10mM stock solution in dimethylsulphoxide. All dilutions were made in a buffer consisting of: 20mM Tris- HCI, 150mM NaCI, 5mM CaCI 2 , 0.1% bovine serum albumin, pH 7.5.
  • the substrate final cone, of 400 ⁇ M
  • Factor Vila final cone, of 10nM in the presence of recombinant soluble tissue factor at optimal concentration
  • compound at appropriate concentrations incubated for 15 minutes and the reaction monitored for 30 minutes at 405nm using a BioTek EL340 plate reader; the assay was performed at 37°C.
  • IC 50 values the data were analyzed using. Kineticalc ® with a 4-parameter curve fitting procedure.
  • the substrate final cone, of 178 ⁇ M
  • chymotrypsin final cone, of 0.2 ⁇ g/mL
  • the reaction monitored for 10 minutes at 405nm using a BioTek EL340 plate reader: the assay was performed at 30°C.
  • IC 50 values the data were analysed using Kineticalc ® with a 4-parameter curve fitting procedure.
  • the compounds were preincubated with chymotrypsin for these times prior to addition of the chromogenic substrate.
  • Compounds of the invention may be tested for their Cathepsin G inhibitory activity as determined in vitro by their ability to inhibit human neutrophil Cathepsin G in a chromogenic assay, using N-succinyl-Ala-Ala-Pro-Phe-p- nitroanilide as the chromogenic substrate.
  • Compounds were diluted from a 10mM stock solution in dimethylsulphoxide. All dilutions were made in a buffer consisting of: 100mM HEPES, 300mM NaCI, pH 7.2. Briefly, the enzyme (1.25ug/mL final ), buffer and compound at appropriate concentrations were incubated for 15 mins at 30°C.
  • the mechanism of binding of the compounds was determined using biophysical techniques such as mass spectrometry and X-ray crystallography. Briefly, crystal structures were prepared in complex with thrombin by soaking and co- crystallisation. X-ray data was collected using a FAST area detector system and difference fourier analysis identified the binding modes of the inhibitors. The bound conformations for each inhibitor were obtained after subsequent refinement cycles which often identified an acylation event.
  • This compound was prepared by the method of Archille Barco et al J.Org.Chem.1992, 57, 6279 as a medium brown oil.
  • Tic cyclohexane:ethyl acetate:3:1) Rf 0.26.
  • step (a) A solution of the product of step (a) (397g) in absolute ethanol (1.41) was reduced at room temperature with stirring over a platinum/carbon catalyst (5% 115g) under a hydrogen atmosphere. The reaction proceeded to completion over 5h (H 2 uptake 99.71). The reaction mixture was filtered through celite and evaporated in vacuo to give the title compound as a light brown oil (303g).
  • step (c) A solution of the product of step (c) (100g) in absolute ethanol (1.251) was reduced over palladium on carbon catalyst (10%, 20g) under a hydrogen atmosphere at room temperature and with stirring (Hydrogen uptake 7.5I). After 20h the reaction mixture was filtered through hyflo and evaporated in vacuo to give the title compound as a colourless oil (71 g) which crystallised to a white solid on standing. Tic (dichloromethane; ethanol; ammonia 80:20:2) Rf 0.4.
  • step (f) The product of step (f) (5g) was dissolved in 4M hydrogen chloride in dioxan (53ml) at room temperature. The solution was stirred for 3h and evaporated. The residue was partitioned between water (100ml) and ether (50ml). The aqueous layer was washed with ether (50ml) and then basified with saturated aqueous sodium hydrogen carbonate (100ml). The mixture was extracted with ethyl acetate (3x50ml). The combined extracts were dried (Na 2 SO 4 ) filtered and evaporated to give the title compound as a clear, pale yellow, viscous oil (3.7g). Mass spec MH + (found) 347 MH + (calc) 347.
  • step (h) rel-(3R,3aS,6aR)-3-Allyl-2-oxo-hexahydro-pyrrolo[3,4-blpyrrole-5-carboxylic acid benzyl ester
  • a solution of the product of step (g) (3.5g) in a mixture of dry tetrahydrofuran (35ml) and dry tetramethylethylenidiamine (35ml) was cooled to 5°C.
  • a solution of t-butyl magnesium chloride (1M, 32ml) in tetrahydrofuran was added over 25 min and the mixture was allowed to warm to room temperature over 1.5h.
  • Oxalyl chloride (4.2ml) was added to a stirred solufion of 4-(piperidin-1-yl)-but-2- enoic acid hydrochloride (10g) in dichloromethane (200ml). Dimethylformamide (2 drops) was then added. The resultant suspension was sfirred for 4h and then concentrated to ⁇ 25ml. The slurry obtained was stirred a further 1h and then filtered. The filter pad was washed with DCM (50ml) and dried in vacuo to give the title compound as a white solid (4.95g). M.p. 120-124°C (decomposition).
  • step (k) (0.44g) was added at room temperature to a solution of the product of step (j) (0.28g) in DCM (20ml) containing solid sodium hydrogen carbonate (0.34g). The reaction was stirred for 4h. The mixture was washed with saturated aqueous sodium hydrogen carbonate (20ml). The aqueous phase was separated and extracted with dichloromethane (20ml). The combined organics were washed with water (2 x 30ml), dried over sodium sulphate and evaporated in vacuo. The resultant gum was purified by flash column chromatography on silica gel (Merck 9385) using gradient elution.
  • Triethylamine (246 ⁇ l) and N-acetylsulfanilyl chloride (108mg) were added to a solution of the product of Compound Example 1 , step (j) (1 OOmg) in dichloromethane (3.3ml). The solution was stirred at room temperature for 5h. Sodium hydrogen carbonate (150mg) was added and the reaction stirred overnight. The resultant suspension was washed with saturated sodium hydrogen carbonate solution. The solvent was removed in vacuo. The resultant solid was purified using pre-packed silica cartridges.
  • step (a) To a solution of the product of step (a) (85g) in ethanol (1500ml) was added potassium hydroxide (15.3g). The resultant mixture was stirred at room temperature for 3.5h and then concentrated under reduced pressure. The residue was then partitioned between sodium carbonate (2N) and diethyl ether. The aqueous layer was acidified to pH2 with concentrated hydrochloric acid and then extracted with diethyl ether. The combined organic extracts were dried (sodium sulfate) and concentrated under reduced pressure to give, after triturating in diethyl ether, the title compound (37g) as a white solid. Analysis: Found: C,27.0; H.3.4; N.10.35% C 6 H 9 IN 2 O 2 requires C.26.9; H.3.4; N, 10.45%
  • step (e) A solution of the product of Compound Example 1 , step (e) (5.2g) in THF (110ml) at -79°C was treated with a THF solution of lithium hexamethyldisilazide (1 M, 60ml) over 20 min. After 2.5h at -76°C, the product of step (b) (4.18g) in THF/HMPA (27ml/35ml) was added over 20 min and the resultant solution was maintained at -70°C for 2. Oh. After reaching 0°C (over 1 h), the reaction was quenched with ammonium chloride solufion and diluted with water.
  • step (d) trans-3-Amino-4-[1 -ethoxycarbonyl-5-(5-oxo-4,5-dihydro-[1 ,2,4]oxadiazol-3- yl)-pentyl]-pyrrolidine-1 -carboxylic acid benzyl ester trifluoro-acetate
  • step (c) was dissolved in trifluoroacetic acid (20ml) and dichloromethane (175ml). After 2h, the solvents were removed under reduced pressure to give the title compound (4.0g) as a yellow oil.
  • step (f) rel-(3R,3aR,6aS)-2-Oxo-3-[4-(5-oxo-4,5-dihydro-[1 ,2,4]oxadiazol-3-yl)-butyl]- hexahydro-pyrrolo[3,4-b]pyrrole-5-carboxylic acid benzyl ester-GR216138X
  • a mixture of the product of step (e) (3.0g), diphenylphosphorylazide (2.8ml) and triethylamine (4.5ml) in DMF (150ml) was stirred at room temperature for 24h. The mixture was concentrated under reduced pressure and the residue subjected to preparative h.p.l.c.
  • reaction was concentrated under reduced pressure and the residue subjected to preparative h.p.l.c. (gradient profile 10-90% (ii) in 25min) to give the title compound (0.018g) as a cream coloured solid by concentration of the required fraction under reduced pressure and drying by repetitive addition of acetonitrile and concentrafion under reduced pressure.
  • Preparative high performance liquid chromatography (h.p.l.c.) was carried out using a Dynamax 60A C18 8 ⁇ M 25cm x 41.4mm i.d. column eluted with a mixture of solvents (i) 0.1% trifluoroacetic acid in water and (ii) 0.05% trifluoroacetic acid in acetonitrile, at a flow rate of 45mi/minute.
  • Analytical h.p.l.c. was carried out using a Dynamax 60A C18 8 ⁇ M 25cm x 4.6mm i.d. column using eluants as for preparative h.p.l.c. at a flow rate of 1 ml/minute.
  • Compound Examples 4-6 were prepared by a method analogous to that for Compound Example 3: Compound Example 4 rel-(3R,3aR,6aS)-5-(1 H-benzoimidazole-2-sulfonyl)-3-(4-carbamimidoyl-butyl)-2- oxo-hexahydro-pyrrolo[3,4-b]pyrrole-1 -carboxylic acid methyl ester trifluoro- acetate Mass spectrum: Found: MH + 463
  • step (a) rel-(3R,3aS,6aS)-3- ⁇ 4-[4-(2-Nitro-benzyl)-5-oxo-4,5-dihydro-[1 ,2,4]oxadiazol- 3-yl]-butyl ⁇ -2-oxo-hexahydro-pyrrolo[3,4-b]pyrrole-5-carboxylic acid benzyl ester-
  • step (f) (0.2g) o- nitrobenzylbromide (0.216g) and triethylamine (0.174ml) in DMF (3ml) was stirred at room temperature for 26h.
  • step (b) A solufion of the product of step (b) (0.15g) in anhydrous dioxan (120ml) was photolysed for 2h, and then concentrated under reduced pressure. The residue was subjected to flash column chromatography eluting with cyclohexane:ethyl acetate: acetic acid (10:90:1) to give the title compound (0.048g) as an orange solid.
  • reaction was concentrated under reduced pressure and the residue subjected to preparative h.p.l.c. (gradient profile 5-50% (ii) in 10min; 50% (ii) isochratic for 5min) to give the title compound (0.007g) as a cream coloured solid by concentration of the required fraction under reduced pressure and drying by repetitive addition of acetonitrile and concentration under reduced pressure.

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Abstract

L'invention concerne un inhibiteur d'une enzyme de sérine protéase qui est un dérivé substitué de trans-hexahydropyrrolo[3,4-b]pyrrol-2-one.
PCT/EP1998/005744 1997-09-09 1998-09-07 Derives de pyrrolopyrrolidine et leur utilisation comme inhibiteurs de serine protease WO1999012935A1 (fr)

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JP2000510742A JP2001515906A (ja) 1997-09-09 1998-09-07 ピロロピロリジン誘導体およびセリンプロテアーゼインヒビターとしてそれらの使用
EP98950011A EP1015456A1 (fr) 1997-09-09 1998-09-07 Derives de pyrrolopyrrolidine et leur utilisation comme inhibiteurs de serine protease
AU96243/98A AU9624398A (en) 1997-09-09 1998-09-07 Pyrrolopyrrolidine derivatives and their use as serine protease inhibitors

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GBGB9719161.3A GB9719161D0 (en) 1997-09-09 1997-09-09 New therapeutic method
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WO2000018770A1 (fr) * 1998-09-30 2000-04-06 Glaxo Group Limited Derives de pyrrolopyrrolone comme agents antiviraux
US6221914B1 (en) 1997-11-10 2001-04-24 Array Biopharma Inc. Sulfonamide bridging compounds that inhibit tryptase activity
US6335324B1 (en) 1998-06-25 2002-01-01 Bristol-Myers Squibb Co. Beta lactam compounds and their use as inhibitors of tryptase
WO2002057273A1 (fr) * 2001-01-20 2002-07-25 Trigen Limited Inhibiteurs de la serine protease comprenant un accepteur de liaison hydrogene
EP1325962A1 (fr) * 2001-12-17 2003-07-09 Gentium S.p.A. Procédé pour la détermination de l'activité biologique de la defibrotide
WO2004094372A3 (fr) * 2003-04-16 2005-06-02 Bristol Myers Squibb Co Biarylmethyl indolines, indoles et tetrahydroquinolines utiles en tant qu'inhibiteurs de la serine protease
US7138412B2 (en) 2003-03-11 2006-11-21 Bristol-Myers Squibb Company Tetrahydroquinoline derivatives useful as serine protease inhibitors
US7417063B2 (en) 2004-04-13 2008-08-26 Bristol-Myers Squibb Company Bicyclic heterocycles useful as serine protease inhibitors
WO2013190582A1 (fr) * 2012-06-22 2013-12-27 Gentium S.P.A. Procédé à base d'euglobuline pour la détermination de l'activité biologique de défibrotide
EP2982668A2 (fr) 2002-12-03 2016-02-10 Pharmacyclics LLC Dérivés de 2-(2-hydroxybiphényl-3-yl)-1h-benzoimidazole-5-carboxamidine en tant qu'inhibiteurs du facteur viia inhibitors pour le traitement de maladies thromboemboliques
US9867843B2 (en) 2010-11-12 2018-01-16 Gentium S.R.L. Defibrotide for use in prophylaxis and/or treatment of graft versus host disease (GVHD)
US10393731B2 (en) 2014-11-27 2019-08-27 Gentium S.R.L. Cellular-based method for determining the biological activity of defibrotide

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WO1995024186A1 (fr) * 1994-03-11 1995-09-14 Pharmacopeia, Inc. Derives de sulfonamide et leurs utilisations
WO1997048706A1 (fr) * 1996-06-18 1997-12-24 Warner-Lambert Company Pyrrolo(1,2-a)pyrazine 1,4-diones servant d'inhibiteurs des serines proteases
WO1998043975A1 (fr) * 1997-04-02 1998-10-08 Glaxo Group Limited Derives de pyrolopyrrolone

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WO1995024186A1 (fr) * 1994-03-11 1995-09-14 Pharmacopeia, Inc. Derives de sulfonamide et leurs utilisations
WO1997048706A1 (fr) * 1996-06-18 1997-12-24 Warner-Lambert Company Pyrrolo(1,2-a)pyrazine 1,4-diones servant d'inhibiteurs des serines proteases
WO1998043975A1 (fr) * 1997-04-02 1998-10-08 Glaxo Group Limited Derives de pyrolopyrrolone

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Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6221914B1 (en) 1997-11-10 2001-04-24 Array Biopharma Inc. Sulfonamide bridging compounds that inhibit tryptase activity
US6335324B1 (en) 1998-06-25 2002-01-01 Bristol-Myers Squibb Co. Beta lactam compounds and their use as inhibitors of tryptase
WO2000018770A1 (fr) * 1998-09-30 2000-04-06 Glaxo Group Limited Derives de pyrrolopyrrolone comme agents antiviraux
WO2002057273A1 (fr) * 2001-01-20 2002-07-25 Trigen Limited Inhibiteurs de la serine protease comprenant un accepteur de liaison hydrogene
AU2002360945B2 (en) * 2001-12-17 2007-11-08 Gentium S.R.L. A method for determining the biological activity of defibrotide
WO2003052130A3 (fr) * 2001-12-17 2004-01-15 Gentium Spa Methode de mesure de l'activite biologique d'un defibrotide
US7338777B2 (en) * 2001-12-17 2008-03-04 Gentium Spa Method for determining the biological activity of defibrotide
CN100408690C (zh) * 2001-12-17 2008-08-06 金蒂姆股份公司 去纤肽生物学活性的测定方法
EP1325962A1 (fr) * 2001-12-17 2003-07-09 Gentium S.p.A. Procédé pour la détermination de l'activité biologique de la defibrotide
KR100912424B1 (ko) * 2001-12-17 2009-08-14 젠티엄 에스피에이 디파이브로타이드의 생물학적 활성 측정 방법
EP2982668A2 (fr) 2002-12-03 2016-02-10 Pharmacyclics LLC Dérivés de 2-(2-hydroxybiphényl-3-yl)-1h-benzoimidazole-5-carboxamidine en tant qu'inhibiteurs du facteur viia inhibitors pour le traitement de maladies thromboemboliques
US7138412B2 (en) 2003-03-11 2006-11-21 Bristol-Myers Squibb Company Tetrahydroquinoline derivatives useful as serine protease inhibitors
US7709646B2 (en) 2003-03-11 2010-05-04 Bristol-Myers Squibb Company Tetrahydroquinoline derivatives useful as serine protease inhibitors
WO2004094372A3 (fr) * 2003-04-16 2005-06-02 Bristol Myers Squibb Co Biarylmethyl indolines, indoles et tetrahydroquinolines utiles en tant qu'inhibiteurs de la serine protease
US7129264B2 (en) 2003-04-16 2006-10-31 Bristol-Myers Squibb Company Biarylmethyl indolines and indoles as antithromboembolic agents
US7417063B2 (en) 2004-04-13 2008-08-26 Bristol-Myers Squibb Company Bicyclic heterocycles useful as serine protease inhibitors
US9867843B2 (en) 2010-11-12 2018-01-16 Gentium S.R.L. Defibrotide for use in prophylaxis and/or treatment of graft versus host disease (GVHD)
WO2013190582A1 (fr) * 2012-06-22 2013-12-27 Gentium S.P.A. Procédé à base d'euglobuline pour la détermination de l'activité biologique de défibrotide
US9902952B2 (en) 2012-06-22 2018-02-27 Gentrum S.R.L. Euglobulin-based method for determining the biological activity of defibrotide
CN110079580A (zh) * 2012-06-22 2019-08-02 真蒂奥姆责任有限公司 用于测定去纤维蛋白多核苷酸的生物活性的基于优球蛋白的方法
US11085043B2 (en) 2012-06-22 2021-08-10 Gentium S.R.L. Euglobulin-based method for determining the biological activity of defibrotide
US11236328B2 (en) 2012-06-22 2022-02-01 Gentium S.R.L. Euglobulin-based method for determining the biological activity of defibrotide
CN110079580B (zh) * 2012-06-22 2022-12-06 真蒂奥姆责任有限公司 用于测定去纤维蛋白多核苷酸的生物活性的基于优球蛋白的方法
US11746348B2 (en) 2012-06-22 2023-09-05 Gentium S.R.L. Euglobulin-based method for determining the biological activity of defibrotide
US10393731B2 (en) 2014-11-27 2019-08-27 Gentium S.R.L. Cellular-based method for determining the biological activity of defibrotide

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